PROJECT SUMMARY The mechanisms underlying the complex process of protein translocation across a plasma membrane have been notoriously difficult to define. Currently, few model systems exist in which this process has been adequately described, with a serious lack of progress being made on this subject in recent years. One of the factors that has led to a lack of development in this area is the absence of structural data which describe key intermediates that occur during protein translocation. This is largely due to complications in obtaining pure, homogenous, samples in these states. With the advent of high-resolution cryo electron microscopy (cryo-EM) and the development of sophisticated data processing algorithms that are designed to tackle these issues, we are now in a position to make significant progress in addressing this question. Under this award, I will describe the fundamental process of protein translocation using a secreted bacterial toxin known as the Clostridioides difficile transferase toxin (CDT) as a model system. The experiments I propose here will build upon my strong foundation of structural and biochemical training as well as the preliminary data that I have collected on this subject, including five distinct structures of CDT that I have already solved by cryo-EM. These structures will be used to guide the development of novel tools and assays to rigorously describe CDT translocation mechanisms. I will address the outstanding questions of how pore formation is regulated and determine what factors drive protein translocation in this model system leading to the elucidation of several structures of key translocation intermediates that exist during this process. These structures will be probed both in vitro and in tissue culture to define protein translocation mechanisms in the context of intoxication. The cell based assays I am proposing will form a crucial component of my one additional year of postdoctoral training under this award as guided by Dr. Borden Lacy during which time I will gain expertise in the maintenance and processing of mammalian tissue culture cell lines for a variety of downstream applications. I will use this base knowledge to build my expertise in confocal microscopy and flow cytometry, taking advantage of the strong research environment at commitment to training at Vanderbilt University Medical Center. The training that I will receive under this award will add a new facet to the independent research program that I intend to develop at a R1 institution. The focus of my lab will be to continue probing the phenomenon of protein translocation mechanisms using bacterial secretion systems as a tool. I believe my extensive structural biology and biochemical background coupled with my extensive experience as a mentor to graduate students make me well suited for such a role.